ABSTRACT The human mouth is colonized by a complex microbial community that plays a key role in human health and disease. Increasingly, it is recognized that the spatial organization of microbiomes is critical to understanding the interactions of the individual taxa that comprise a community. Although next-generation DNA sequencing technology and metagenomics have revolutionized the analysis of microbial communities, these technologies require that the microbial cells first be broken open and the DNA extracted, processes in which spatial organization is lost. Thus, a major gap in our understanding is the lack of information at the spatial scale at which microbiomes live and work--the micron scale. In our previous grant period, we sought to fill this gap by using the strategy of combinatorial labeling and spectral imaging fluorescence in situ hybridization (CLASI-FISH) to analyze the micron-scale architecture of microbial communities in the healthy oral cavity--basically to determine ?who lives next to who? and ?who lives next to what?. This strategy was successful in that it revealed highly organized and hitherto unanticipated microbial community structures at the genus level in dental plaque and on the tongue dorsum. Much work remains to be done in characterizing these communities and ones at other oral sites as well. But it is clear that specification of community structure must be deepened to the species level. Further, it is clear that one needs to go beyond taxonomic identification. To understand the mechanistic bases of microbial relationships, it is necessary to gain data on their functional expression at the single-cell level. Also, the static images acquired after imaging fixed cells provide only snapshots in time. It is necessary to figure out ways to study the dynamics of microbial communities?how they form, develop and maintain themselves. The specific aims of this proposal are directed at these three issues. We will broaden and deepen analysis of key oral habitats so that the major microbial taxa within oral communities can be identified to species level. To test hypotheses generated from the structural results, we will develop probes for expression of key mRNA molecules at the single-cell level. To test hypotheses on the development and dynamics of microbial communities, we will develop probes for metabolic capacity in living cells using bioorthogonal click chemistry and combine their application with culture of oral microcosms. The three approaches build on the work of the previous grant period, sharing the common thread of single-cell analysis through multiplexed imaging. By revealing the precise, micron-scale structure, mRNA expression and dynamics of the oral microbiome, this research will address fundamental mechanisms and principles of community function and assembly. It will impact the broader oral and microbial research communities by developing methods, analysis tools and probes, and it should help identify novel targets for health maintenance or therapeutic intervention. ! !